Identification, quantification, and isolation of low abundance proteins from complex mixtures is, at best, a difficult task. This is especially the case for proteins carrying a post-translational modification (PTM) that affects their half-life or regulatory properties. Examples of such PTMs include phosphorylation, glycosylation (especially O-GlcNAcylation), and ubiquitinylation. In many instances, one PTM can abrogate or enhance other PTMs on the same protein providing exquisite mechanisms for control of the protein's activity. Fishing a protein with one particular PTM out of the pool of possible modifie proteins becomes nearly impossible without selective tools. A further complication in the case of ubiquitinylation is the presence of multiple types of Ub-Ub linkages in polyubiquitin chains. Ubiquitin (Ub) is attached, via isopeptide bonds, to lysine residues in the target protein. These Ub-moieties can then serve as substrates for the conjugation of additional Ubs, again through the formation of isopeptide bonds between the C- terminus of one Ub and any of seven (7) lysines in the target Ub. The general consensus in the field is that chains with different linkages convey different meanings to the cell and hence, determine the ultimate fate of the protein, be it degradation, translocation, phosphorylation, etc. The precise information encoded in different chain linkages is largely unknown due to the lack of specific reagents that recognize different linkages. The goal of this proposal is to develop tools that allow the selective identification, quantification, and isolation of proteins modified by polyubiquitin chains containing different linkages. This will be accomplished using information encoded in the human genome that allows the cell to discriminate between different linkages, i.e. Ub-binding domains (UbDs). In Phase I, we will identify and characterize novel UbDs exhibiting, at least, partial selectivity. In Phase II we will use these UbDs to construct higher avidity reagents capable of linkage-specific discrimination. Both ubiquitinylation and de- ubiquitinylation have been linked to cancer, inflammation and neurological diseases; hence, the tools developed in this grant will have a major impact on our ability to dissect these disease processes.